The diagnosis of Parkinson disease (PD) and other alpha-synucleinopathies is clinical and often uncertain in early, mild cases. We hypothesized that cerebrospinal fluid (CSF) levels of neuronal metabolites of catecholamines would detect central catecholamine deficiency and thereby help diagnose these disorders. In a large study we measured CSF levels of catechols including dopamine, norepinephrine, and their main respective neuronal metabolites dihydroxyphenylacetic acid (DOPAC) and dihydroxyphenylglycol (DHPG) in PD and two other synucleinopathies, multiple system atrophy (MSA) and pure autonomic failure (PAF). The PD, MSA, and PAF groups all had lower CSF DOPAC and DHPG levels than did the controls. DOPAC was lower in PD than in PAF and DHPG lower in PAF than in PD. CSF DOPAC was 100% sensitive at 89% specificity in separating patients with recent onset of Parkinsonism from controls. From these findings we concluded that synucleinopathies feature CSF neurochemical evidence for central dopamine and norepinephrine deficiency, that PD and PAF involve differential dopaminergic vs. noradrenergic lesions, and that CSF DOPAC provides a sensitive clinical laboratory means to identify PD, even early in the disease. We are testing neuroimaging and neurochemical putative biomarkers of presymptomatic PD (Goldstein DS, Holmes C, Sewell L, Park M-Y, Sharabi Y. Sympathetic noradrenergic before striatal dopaminergic denervation: Relevance to Braak staging of synucleinopathy. Clin Auton Res 2011 Jul 28. Epub ahead of print). Under Protocol 09-N-0010 we have so far screened 45 eligible people to confirm their risk factors and admitted 14 for biomarkers testing. Of the 14, by the time of inpatient testing 1 already had PD diagnosed and was excluded from further participation. CSF catechols data are available from 12 other subjects;CSF DOPAC and DOPAC:dopamine have already been found to be significantly decreased compared to volunteers without multiple risk factors. Considered with the results of CSF biomarkers testing noted above in patients with diagnosed PD, low CSF DOPAC and DOPAC:dopamine ratios seem highly promising neurochemical biomarkers of central dopamine deficiency and presymptomatic PD. CSF levels of DOPAC and especially of dopamine are extremely low. We compared liquid chromatography and triple quadrupole mass spectroscopy (LC/MS/MS) vs. liquid chromatography and electrochemical detection (LCED) for high-sensitivity measurements of catecholamines in human biological fluids such as plasma. So far LCED has proven more sensitive and more reliable than LC/MS/MS for detection of low levels of catecholamines such as dopamine. Ideally, validation of neurochemical or neuroimaging as diagnostic biomarkers of PD and related disorders should include post-mortem confirmation. We evaluated a patient who had MSA clinically based on prominent early OH, rapid progression of Parkinsonism, and lack of response to dopaminergic drugs. Clinical laboratory evaluation, however, showed neuroimaging evidence of cardiac sympathetic denervation by 18F-dopamine PET scanning and severely decreased tyrosine hydroxylase immunostaining in arrector pili muscle in skin biopsy samples, a combination that supported a diagnosis of PD+OH. Post-mortem neuropathology showed substantia nigra Lewy bodies diagnostic of PD and no evidence of MSA. Catechol neurochemistry revealed profoundly decreased putamen dopamine, as expected in PD, and a low myocardial concentration of norepinephrine. The latter provided the first neurochemical confirmation of the validity of 18F-dopamine PET scanning to detect cardiac sympathetic denervation. Lewy neurites containing precipitated alpha-synuclein were found in the cervical sympathetic trunk, demonstrating synucleinopathy not only in the brain but also in the autonomic nervous system in this patient. In collaboration with J. Zhang, of the Univ. of Washington, we are exploring CSF proteomic biomarkers of PD, MSA, and PAF. Phosphorylated alpha-synuclein (PS-129), a protein critically involved in the pathogenesis of PD, was measured, along with total synuclein, in patients with PD, other neurodegenerative disorders, and controls. CSF PS-129 levels correlated with PD severity and when combined with total synuclein provided high sensitivity and specificity for differential diagnosis among clinical overlapping parkinsonian disorders. Decreased CSF DJ-1 or alpha-synuclein is a potential index for PD diagnosis. Also in collaboration with J. Zhang, total tau, phosphorylated tau, amyloid beta peptide 142 (Ab142), Flt3 ligand, and fractalkine levels were measured in CSF in a large cohort of PD patients at different stages as well as healthy and diseased controls. The results demonstrated that with CSF Flt3 ligand, PD could be clearly differentiated from MSA (Wang et al., Am J Pathol 2011;178:1509-1516). In another collaborative study with Dr. Zhang we examined other CSF biomarkers that might distinguish PD from MSA. Complement activation, a key component of neuroinflammation, has been reported in both PD and Alzheimer's disease (AD);however, it has been unclear whether complement activation and neuroinflammation in general are different from each another in major neurodegenerative disorders. In this study, CSF complement 3 (C3) and factor H (FH) were measured and evaluated together with amyloid-beta 42 (Abeta42). The C3/FH ratio demonstrated high sensitivity and specificity in differentiating patients with MSA from those with AD or PD and control individuals (Shi et al., Ann Neurol 2011;69:570-580). Given our findings indicating clear abnormalities of CSF levels of catechols (especially DOPAC) in PD, and the findings from the collaborative studies with Dr. Zhang about CSF proteomic biomarkers, we plan on extending the collaboration to include measurements both of CSF catechols and proteins of interest (e.g., PS-129), in the hope that these will provided the long sought for CSF biomarker pattern that can diagnose PD in individual patients. Until recently, no radioligand for neuroimaging successfully and specifically visualized central neural sites of noradrenergic innervation. 11C-Methylreboxetine (11C-MRB) has these capabilities. Recent studies show that in humans, after 11C-MRB administration positron emission tomographic (PET) scanning with a High Resolution Research Tomograph (HRRT) can visualize the locus ceruleus, the pontine cluster of neurons that is the main source of norepinephrine in the brain. Dr. Y-S Ding of the Yale PET Center is the leader in this area and has agreed to collaborate with the Clinical Neurocardiology Section (CNCS) and introduce 11C-MRB PET scanning at the NIH Clinical Center. We plan initially to validate and apply the technology in patients with PD or PAF, conditions that from a neurochemical point of view involve central norepinephrine deficiency. An Amendment to NIH Clinical Protocol 03-N-0004 has been submitted, under which we will take the lead in developing clinical 11C-MRB scanning in PD and related disorders.